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Role of Molecular Chaperones in Biogenesis of the Protein Kinome

  • Atin K. Mandal
  • Maria A. Theodoraki
  • Nadinath B. Nillegoda
  • Avrom J. CaplanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 787)

Abstract

Molecular chaperones promote polypeptide folding in cells by protecting newly made and otherwise misfolded proteins against aggregation or degradation by the ubiquitin proteasome pathway. The roles of Saccharomyces cerevisiae Cdc37 and Ydj1 molecular chaperones are described in this chapter. We focus on biogenesis of protein kinases that require several different molecular chaperones for their proper folding. Specific among these is Cdc37, which binds directly to its kinase clients either during or shortly after translation and protects them against rapid proteasomal degradation. Ydj1 has a similar role, but is less specific for protein kinases in its role as a molecular chaperone. The method that we describe uses pulse chase and immunoprecipitation to analyze the fate of newly made proteins. Two kinetically distinct pathways of degradation can be discerned using this methodology that is dependent on the presence of an Hsp90 inhibitor or occurs in mutants of the molecular chaperones under study. The first is “zero-point” degradation that occurs either during or immediately after translation. The second is a slower pathway, where the half-life of kinase is approximately 20 min after translation.

Key words

Molecular chaperone Protein folding Protein kinase Ubiquitin proteasome system Quality control 

Notes

Acknowledgments

This work was supported by NIH grants RO1GM70596, U54CA132378 (to A.J.C.), and NCRR 5 G12-RR03060 (CCNY).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Atin K. Mandal
    • 1
  • Maria A. Theodoraki
    • 1
  • Nadinath B. Nillegoda
    • 1
  • Avrom J. Caplan
    • 2
    Email author
  1. 1.Department of BiologyCity College New YorkNew YorkUSA
  2. 2.Department of BiologyThe City College of New YorkNew YorkUSA

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